Two 18‐norspirostane steroidal saponins as novel mitophagy enhancers improve Alzheimer's disease

Abstract

The hallmark of Alzheimer's disease (AD) is the progressive accumulation of misfolded proteins, specifically beta-amyloid (Aβ) and Tau, leading to neurotoxicity and impaired neuronal function.1 Impaired mitophagy in AD contributes to protein buildup and subsequent neuronal damage.2 Mitophagy enhancers are investigated as potential therapeutic interventions for AD.3, 4 Deoxytrillenoside CA (DTCA) and epitrillenoside CA (ETCA), two newly discovered compounds derived from Trillium tschonoskii Maxim. (TTM) (Figure 1A), have shown antioxidative properties by inducing autophagy.5 Nevertheless, their impact on mitophagy induction and clearance of AD-related proteins remains unexplored. In this study, our primary focus was to investigate the autophagic degradation effects of DTCA&ETCA on AD-related proteins. To begin, the Aβ fibrillization was measured using the Thioflavin T (ThT) reagent. The results showed that DTCA&ETC, administrated at relatively safe concentrations (Figure S1), exhibited a dose-dependent reduction in ThT fluorescence, indicating the inhibition of Aβ1-42 (Figure 1B,C) and Aβ25-35 fibrilization (Figure S2A,B). The biolayer interferometry (BLI) analysis displayed that DTCA&ETCA directly bound to Aβ1-42 (Figure 1D,E). Subsequently, the effect of DTCA&ETCA on Aβ’s cytotoxicity was examined, revealing an increase in cell viability and a decrease in cell death and apoptosis of a immortalized mouse hippocampal neuronal cell line (HT-22) or a rat adrenal pheochromocytoma cell line (PC-12) cells (Figure 1F-J, Figure S2C-I, S3). To investigate how DTCA&ETCA influence the removal of AD-associated proteins, the green fluorescent protein (GFP) fluorescence intensity and protein expression in neuronal cell lines transfected with enhanced green fluorescent protein fused amyloid precursor protein (EGFP-fused APP), Tau, or Tau P301L plasmid were determined. Figure 1K-U and Figure S4-6 clearly demonstrated that DTCA&ETCA significantly hinder the GFP intensity and protein expression, while not affecting transfection efficacy. Furthermore, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) results indicated that DTCA&ETCA enhanced the viability of transfected neuronal cells (Figure S7). Moreover, the flow cytometry analysis revealed that DTCA&ETCA not only decreased GFP intensity but also reduced the proportion of propidium iodide (PI)-positive cells (Figure S8). Collectively, DTCA&ETCA effectively inhibits Aβ production and facilitates the clearance of AD-related proteins. Autophagy activity was assessed by examining the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3), the number of LC3 puncta, and autophagy flux.5, 6 DTCA&ETCA increased LC3 conversion and puncta in a dose- and time-dependent fashion (Figure 2A-C, Figure S9). Furthermore, DTCA&ETCA induced autophagy flux as indicated by the elevated red fluorescent protein-LC3 (RFP-LC3)/GFP-LC3 ratio (Figure 2D,E). Notably, autophagy flux induced by DTCA&ETCA was impeded by 3-methyladenine (3-MA) and bafilomycin A1 (Baf), demonstrated by reduced LC3 conversion and puncta during autophagy initiation and increased LC3 conversion and puncta during autophagosome-lysosome fusion (Figure 2F, Figure S10). The transmission electron microscopy images revealed increased engulfment of mitochondria (mt) by autophagosomes (av) in HT-22 cells treated with DTCA, ETCA, or urolithin A (UA, a positive mitophagy activator), suggesting the potential induction of mitophagy by DTCA&ETCA (Figure 2G). Mechanistic investigations indicated that DTCA&ETCA inhibited mTOR and activated the AMPK/ULK1 and PINK1/Parkin pathways (Figure 2H, Figure S11). Cotreatment with compound C (CC) and SBI0206965 (SBI) attenuated LC3 conversion and GFP-LC3 puncta induced by DTCA&ETCA (Figure S12). Moreover, SBI significantly reversed the elevated colocalization of GFP-LC3 with mitochondria and the reduced GFP/RFP ratio in DTCA&ETCA-treated HT-22 cells transfected with Mito-QC, a construct expressing a mCherry-GFP tag attached to the outer mitochodrial membrane FIS1 (residues 101-152) (Figure 2I-L). To investigate whether DTCA&ETCA degrade AD-associated proteins via mitophagy, 3-MA and Baf were employed. Figure 2M-O and Figure S13-14 demonstrated that 3-MA and Baf abolished the reduction in GFP expression induced by DTCA and ETCA in HT-22 cells transfected with EGFP-fused APP, Tau or Tau P301L plasmid. Additionally, MEF cells with or without the Atg7 gene were utilized to explore how Atg7 regulates the clearance of AD-associated proteins by DTCA&ETCA. Figure S15 showed that DTCA&ETCA activated autophagy and significantly reduced GFP intensity in wild-type MEFs, while this effect is absent in Atg7-deficient MEFs. Furthermore, DTCA&ETCA inhibited cell apoptosis in EGFP-N1-APP- or EGFP-Tau-P301L-transfected HT-22 cells (Figure S16). Overall, DTCA&ETCA promote the autophagic degradation of AD-related proteins, leading to the alleviation of neuronal apoptosis. To investigate the potential of DTCA&ETCA in stimulating mitophagy in vivo, BC12921, DA2123 and IR1631 strains were utilized.7 Figure 3A-D demonstrated that DTCA&ETCA resulted in an elevation of GFP-LGG-1 foci in DA2123 worms and a decline in p62-GFP protein expression in BC12921 worms. Additionally, DTCA&ETCA significantly decreased the GFP/DsRed ratio in IR1631 worms, implicating the involvement of unc-51 and pdr-1 genes (Figure 3E,F). These findings collectively suggest that DTCA&ETCA induce mitophagy in Caenorhabditis elegans. Subsequently, CL2331, CL4176 and BR5270 strains were employed to investigate the anti-AD effects of DTCA&ETCA.8 Figure 3G,H demonstrated that DTCAT&ETCA significantly reduced Aβ aggregations in the anterior region of CL2331worms. Moreover, in CL4176 worms, DTCA&ETCA exhibited a delay in Aβ-induced paralysis (Figure 3G,I), while in BR5270 worms, it alleviated the food-searching deficit (Figure 3J). When we fed CL4176 worms with RNAi unc-51, RNAi Pdr-1, RNAi bec-1 and RNAi vps-34 bacteria, the paralysis got worse and the delay effect of DTCA&ETCA on paralysis disappeared (Figure 3K,L). These results strongly support the notion that DTCA&ETCA exert potent anti-AD effects by inducing mitophagy in C. elegans. Subsequently, we studied the impact of ETCA on AD using APP/PS1 mice. The result showed that the cognition examined by Morris Water Maze was greatly alleviated by ETCA (Figure 4A-D). Furthermore, ETCA significantly reduced Aβ and p-Tau levels, the Bax/Bcl-2 ratio, GFAP, and Iba-1 contents, while increasing NeuN-positive cells in the brain tissue (Figure 4E-I, Figure S17A,B). Mechanistically, ETCA significantly increased the phosphorylation (p) of AMPK, p-ULK1(Ser555), LC3-II, Parkin and PINK1, while down-regulating p-mTOR, p-ULK (Ser757) and p-70S6K in the brain tissue (Figure 4J-L, Figure S17C,D). Collectively, ETCA improves cognition and pathology of APP/PS1 mice potentially via mitophagy induction. In summary, DTCA&ETCA demonstrate potential as mitophagy enhancers, clearing AD-related proteins through mTOR, AMPK/ULK1 and PINK1/Parkin signaling pathways (Figure S18). These findings enhance our understanding of DTCA&ETCA's therapeutic potential in AD treatment and validate their future clinical application. Additionally, to provide a comprehensive assessment, we intend to investigate the impact of DTCA and ETCA on toxic forms of Aβ and p-tau oligomers in future experiments. This study was supported by Grants from the National Natural Science Foundation of China (grant numbers: 81960787 and 81903829), the Sichuan Science and Technology Program (grant numbers: 2022YFS0620, 2021YJ0180, and 2022YFH0115), the Macao Science and Technology Development Fund of Macao SAR (grant numbers: SKL-QRCM(MUST)−2020-2022 and MUST-SKL-2021-005), Luzhou Science and Technology Project (grant number: 2022-SYF-73), and Southwest Medical University (grant numbers: 2021ZKZD015, 2021ZKZD018, and 2021ZKMS046). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All data generated or analyzed during this study are included in this published article and its supplementary information files. 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